Method for purification of recombinant proteins

ABSTRACT

Purification of poly-amino acid-tagged recombinant proteins has been improved by the use of a carboxymethylated aspartate ligand complexed with a third-block transition metal having an oxidation state of 2 +   and a coordination number of 6. A method for synthesizing the metal ion-CM-Asp complex is also described. Further, the metal ion-CM-Asp complex can be used for screening protein function.

BACKGROUND OF THE INVENTION

Immobilized metal ion affinity chromatography (IMAC) was firstintroduced by Porath (Porath, J., J. Carlsson, I. Olsson, G. Belfrage1975! Nature 258:598-599.) under the term metal chelate chromatographyand has been previously reviewed in several articles (Porath, J. 1992!Protein Purification and Expression 3:263-281; and articles citedtherein). The IMAC purification process is based on the employment of achelating matrix loaded with soft metal ions such as Cu²⁺ and Ni²⁺.Electron-donating groups on the surface of proteins, especially theimidazole side chain of histidine, can bind to the non-coordinated sitesof the loaded metal. The interaction between the electron donor groupwith the metal can be made reversible by lowering the pH or bydisplacement with imidazole. Thus, a protein possessingelectron-donating groups such as histidine can be purified by reversiblemetal complex/protein interactions.

Several different metal chelating ligands have been employed in IMAC topurify proteins. Iminodiacetic acid (IDA) ligand is a tridentate andthus anchors the metal with only three coordination sites (Porath, J.,B. Olin 1983! Biochemistry 22:1621-1630). Because of the weak anchoringof the metal, metal leakage has been known to occur. Thetris(carboxymethyl)ethylenediamine (TED) ligand is pentadentate andforms a very strong metal-chelator complex. The disadvantage of this isthat proteins are bound very weakly since only one valence is left forprotein interaction. Nitrilo triacetic acid (NTA) is a tetradentateligand which attempts to balance the metal anchoring strength withmetal-ion protein interaction properties (Hochuli, E., H. Dobeli, A.Schacher 1987! J. Chromatography 411:177-184). Other chelating ligandshave been reported and are mentioned. See, e.g. Porath (1992), supra.However, these ligands also have certain disadvantages, includingdecreased bonding capacity, decreased specificity, and increased metalleakage.

In 1991, Ford et al. (Ford, C., I. Suominen, C. Glatz 1991! ProteinExpression and Purification 2:95-107) described protein purificationusing IMAC technology (Ni-NTA ligand) as applied to recombinant proteinshaving tails with histidine residues (polyhistidine recombinantproteins). This method takes advantage of the fact that two or morehistidine residues can cooperate to form very strong metal ioncomplexes. The NTA chelating ligand immobilized on agarose and loadedwith Ni²⁺ has been useful in this method (Hochuli et al., supra; U.S.Pat. No. 5,047,513). It is available commercially through Qiagen, Inc.(Chatsworth, Calif.). However, this resin has the disadvantage that theinterchanges between metal ions and poly-histidine recombinant proteinsare not optimal. Metal leakage can occur, and background proteins cansometimes contaminate purification of recombinant proteins.

A metal chelating gel, i.e., carboxymethylated aspartate (CM-Asp)agarose complexed with calcium, has been used for purifying nativecalcium-binding proteins (Mantovaara, T., H. Pertoft, J. Porath 1989!Biotechnology and Applied Biochemistry 11:564-570; Mantovaara, T., H.Pertoft, J. Porath 1991! Biotechnology and Applied Biochemistry13:315-322; Mantovaara, T., H. Pertoft, J. Porath 1991! Biotechnologyand Applied Biochemistry 13:120-126). However, the Ca²⁺ -CM-Asp complexdescribed by Mantovaara et al. has among its disadvantages that it doesnot bind strongly to histidine-tagged recombinant proteins. Anotherdisadvantage, in addition to this inferior binding property, is itsnon-selectivity for histidine tags.

By contrast, the subject invention comprises the CM-Asp chelating ligandcomplexed to a transition metal in an octahedral geometry (coordinationnumber of 6). In this unique configuration, the metal complex can beadvantageously suited for purification of poly-histidine fusedrecombinant proteins. This is a novel use of the CM-Asp ligand and ispart of the subject of the invention herein described.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns a novel IMAC purification method whichemploys immobilized carboxymethylated aspartate (CM-Asp) ligandsspecifically designed for purification of recombinant proteins fusedwith poly-histidine tags. The new purification method is based upon theCM-Asp chelating matrix having the following structure: ##STR1##

A general description of the matrix used in the invention andillustrated above is:

M=transition metal ion in a 2⁺ oxidation state with a coordinationnumber of 6;

R₁ =a linking arm connecting the nitrogen atom of CM-Asp with R₂ ;

R₂ =a functional linking group through which CM-Asp linking arm R₁ isconnected to R₃ ;

R₃ =a polymer matrix, e.g., those polymer matrices typically used inaffinity or gel chromatography.

In a preferred embodiment:

M=Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺ ;

R₁ =--CH₂ CH(OH)CH₂ -- or --CH₂ (OH)CH₂ --O--CH₂ CH(OH)CH₂ --;

R₂ =O, S, or NH; and

R₃ =agarose.

In a particularly preferred embodiment:

M=Co²⁺ ;

R₁ =CH₂ CH(OH)CH₂ ;

R₂ =O; and

R₃ =agarose, cross-linked.

Prior to loading the 6XHis recombinant protein to the resin, recombinantcells can be lysed and sonicated. The lysate can then be equilibratedwith an aqueous buffer (pH 8) which itself does not form chelates withthe metal. An example of an aqueous that can be used at this step in thedescribed procedure is 50 mM sodium phosphate (pH 8.0)/10 mM Tris-HCl(pH 8.0)/100 mM NaCl, or the like. The equilibration buffer can containdenaturing agents or detergents, e.g., 10% "TRITON X-100," 6Mguanidinium HCl, or the like. After binding the prepared 6XHisrecombinant protein on the metal CM-Asp chelating resin (the "CM-Aspresin complex"), the protein-bound resin is washed at pH 7.0 or 8.0. Theelution of the protein can be carried out at a constant pH or with adescending pH gradient. In a preferred embodiment, protein elution canbe achieved at a pH of about 6.0 to about 6.3. Alternatively, the 6XHisrecombinant protein bound to the CM-Asp chelating resin can be washedwith low concentrations (less than 100 mM) of imidazole at pH 8.0 andthen eluted by increasing the imidazole concentration to 40-100 mM.

Also included as an aspect of the subject invention is a scaled-upsynthesis of the CM-Asp derivatized agarose chelating resin. It is animproved version of a previously reported small scale preparation(Mantovaara, T., H. Pertoft, J. Porath 1991! Biotechnology and AppliedBiochemistry 13:315-322). The improvement includes particular conditionsfor oxirane-agarose formation, temperature controlled conjugation ofaspartic acid to the oxirane-agarose, and high ionic strength washing toremove extraneously bound metals. These conditions, temperatures, andionic concentrations are described in detail herein.

An additional application of the subject invention includes screeningfor protein function on a microtiter plate or filter. The additionalapplications for the subject invention also include protein-proteininteraction studies, as well as antibody and antigen purification. Forexample, by immobilization of the Co²⁺ moiety onto 96-well plates byCM-Asp, such plates can be used for quantitation of 6XHistidine-taggedprotein, protein-protein interaction studies, diagnostic screening fordiseases, antibody screening, antagonist and agonist screening fordrugs, and reporter gene assays. Co²⁺ can also be immobilized onto amembrane, e.g., a nylon membrane, by CM-Asp, which can be used to liftproteins from expression libraries to make protein libraries from cells.The membranes also can be used for screening of engineered enzymes.Application of the subject invention can also be extended topurification of any interacting molecule, e.g., nucleic acids or smallco-factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an outline illustrating a process for purifying recombinant6XHis protein using CM-Asp chelating resin.

FIGS. 2A-2B show a comparison of Co²⁺ CM-Asp chelating resin with Ni-NTAon 6XHis prepro-α-factor purification under denaturing conditions usingpH gradient.

Legend for FIGS. 2A-2B: lane 1: crude lysate; lane 2: flowthrough; lane3: washed with 6M Gu-HCl, 0.1M NaH₂ PO₄, pH 8.0; lane 4: washed with 8Murea, 0.1M NaH₂ PO₄ ; lane 5: washed with 8M urea, 0.1M NaH₂ PO₄, pH8.0; lane 6: eluted with 8M urea, 0.1M NaH₂ PO₄, pH 6.3; lane 7: elutedwith 8M urea, 0.1M NaH₂ PO₄, pH 6.3; lane 8: eluted with 8M urea, 0.1MNaH₂ PO₄, pH 6.3; lane 9: eluted with 8M urea, 0.1M NaH₂ PO₄, pH 5.9;lane 10: eluted with 8M urea, 0.1M NaH₂ PO₄, pH 4.5; lane 11: elutedwith 6M Gu-HCl, 0.1M NaH₂ PO₄, 0.2M acetic acid; lane M: MW sizemarkers.

FIG. 3 shows 6XHis tagged DHFR purification by Co²⁺ CM-Asp chelatingresin under native conditions. Legend: Lane 1: clarified lysate; lane 2:flowthrough; lane 3: first wash; lane 4: third wash; lane 5: DHFR finalelution.

FIG. 4 shows 6XHis tagged DHFR purification by Co²⁺ CM-Asp chelatingresin under denaturing conditions. Legend: Lane 1: clarified lysate;lane 2: flowthrough; lane 3: first pH 7.0 wash; lane 4: second pH 7.0wash; lane 5: DHFR, first pH 6.0 elution; lane 6: DHFR, second pH 6.0elution.

FIG. 5 shows 6XHis tagged DHFR purification by Co²⁺ CM-Asp chelatingresin under native conditions with increasing concentrations ofβ-mercaptoethanol. Legend: lane 1: 20 μl of cell lysate; lanes 2, 4, 6,and 8: 20 μl of flowthrough; lanes 3, 5, 7, and 9: 5 μl of eluant.

FIG. 6 shows yields of 6XHis DHFR from cell lysates purified by Co²⁺CM-Asp chelating resin versus Ni-NTA in the presence ofβ-mercaptoethanol. Protein concentrations were determined by Bradfordassay. Yields are expressed as a percentage of total protein in the celllysate.

FIGS. 7A-7B show purification of 6XHis GFP by Co²⁺ CM-Asp chelatingresin under native conditions. The GFP bands were detected usingClontech's chemiluminescence Western Exposure Kit and overnight exposureto x-ray film.

Legend: lane 1: clarified lysate; lane 2: flowthrough; lane 3: firstwash; lane 4: first elution; lane 5: second elution; lane 6: thirdelution; lane 7: fourth elution.

FIG. 8 shows biological activity of 6XHis GFP purified by Co²⁺ CM-Aspchelating resin. Legend: tube 1: cell lysate; tube 3: flowthrough; tube3: wash; tube 4: first elution; tube 5: second elution; tube 6: thirdelution.

DETAILED DESCRIPTION OF THE INVENTION

The subject method, which employs a CM-Asp metal chelating complex, canadvantageously be used for purification of recombinant proteins having apolyhistidine tail or "tag."

According to one embodiment of the subject invention, a resin ligand,e.g., CM-Asp, is complexed to a metal other than Ca²⁺, forming aCM-Asp-metal complex. Preferably, the CM-Asp ligand used in the subjectinvention is complexed with one of the transition metals (known as athird-block transition metal), e.g., Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺ inan octahedral geometry. Other polymer matrices, e.g., polystyrene (as inmicrotiter plates), nylon (as in nylon filters), or the like, can beapplicable to the subject invention and would readily be recognized bypersons of ordinary skill in the art. The poly-histidine tag possesses"neighboring" histidine residues which can advantageously allow therecombinant protein to bind to these transition metals in a cooperativemanner to form very strong metal ion complexes. This cooperative bindingrefers to what is commonly known in the art as a "neighboring histidineeffect." For purposes of the subject invention, and as would beunderstood by a person of ordinary skill in the art, a "strong" or "verystrong" metal ion complex refers to the bond strength between the metalion and the chelating ligand. A strong or very strong metal ion complex,for example, allows little or essentially no metal leakage from thecomplex so that the purified protein, e.g., a recombinant protein havinga polyhistidine tag, is not contaminated with undesired or "background"protein from a mixture being purified.

The CM-Asp metal complex offers two available valencies that can formstrong and reversible metal complexes with two adjacent histidineresidues on the surface of the recombinant protein. Another advantage tousing the CM-Asp ligand is its ability to strongly anchor the metal ionwhereby metal ion leaking can be virtually eliminated compared to metalleakage observed for other complex binding agents, e.g., Ni-NTA. In amore preferred embodiment, Co²⁺ can be used as the transition metal withCM-Asp. The Co²⁺ -CM-Asp can be less sensitive to reducing agents, suchas β-mercaptoethanol. Metal ion leakage has been shown to remain low,even negligible, in the presence of up to 30 mM β-mercaptoethanol.

One embodiment of the purification process of the subject invention isas follows:

1. Prepare lysate/sonicate containing recombinant 6XHis proteinaccording to standard procedures and techniques well known in the art.

2. Bind 6XHis protein onto metal-loaded CM-Asp chelating resin atslightly basic pH, e.g., about pH 8.0.

3. Wash protein/resin complex at the same basic pH (about pH 8.0).Optional washes at a pH of about 7.0 or with imidazole additive can alsobe included.

4. Elute pure recombinant 6XHis protein with an elution buffer having apH of about 6.0-6.3 or, in the alternative, an elution buffer having apH of about 8.0, plus about 40 to about 100 mM imidazole. The stepsinvolved in a preferred embodiment of the purification process of thesubject invention are illustrated in FIG. 1. The subject process can beemployed batchwise, in spin columns, and in large-scale continuous-flowcolumns.

Buffers used in the above procedures are standard buffers typically usedin similar procedures, with appropriate adjustments and modificationsmade as understood in the art. For example, a high ionic strengthbuffer, e.g., 50 mM phosphate/10 mM Tris/100 mM NaCl can be used, withthe pH adjusted as needed. The phosphate salt component can range from aconcentration of 10-100 mM; Tris from 5-25 mM; and NaCl from 50-200 mM.

Optimal elution conditions depend on the type of impurities, the amountof protein to be purified, and unique properties of the protein, and aredetermined on a case-by-case basis as would be readily recognized by aperson of ordinary skill in the art.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1

Large-Scale Preparation of CM-Asp Chelating Resin

Sepharose CL-6B or CL-6B (Pharmacia, 8.0 L) is washed with ddH₂ O,suction dried, and transferred to a 22-L round bottom flask equippedwith mechanical stirring. Epichlorohydrin (about 2.0 L) is added, theSepharose resin mixed to a thick suspension, and allowed to stand atroom temperature for about 20 minutes. A solution of sodium hydroxide(about 560 g) and sodium borohydride (about 48 g) in approximately 6400mL ddH₂ O is added and the mixture stirred overnight at ambienttemperature. The oxirane-derivatized resin, collected by filtration, iswashed ten times with ddH₂ O (about 10 L each), once with 10% sodiumcarbonate (about 10 L), suction dried, and transferred to a 22-L roundbottom flask. A specimen of the oxirane derivatized resin treated with1.3M sodium thiosulfate is titrated to approximately pH 7.0 to determinethe oxirane concentration (preferably, >700 μmol/g.).

To a solution of sodium hydroxide (approximately 268 g) in about 7.6 LddH₂ O is added L-aspartic acid (about 575 g) and sodium carbonate(about 1700 g), keeping the temperature below about 25° C. The pH isadjusted to approximately 11.0 and the solution added to the resin.Using mechanical stirring and a heating mantle, the reaction mixture isbrought to about 80° C. for 4 hours and allowed to cool to roomtemperature overnight. The resin was collected by filtration, washed tentimes with ddH₂ O (about 10 L each), once with 10% sodium carbonate(about 10 L), suction dried, and transferred to a 22-L round bottomflask equipped with mechanical stirring.

To an ice-cooled solution of sodium hydroxide (about 900 g) in 12 L ddH₂O was added bromoacetic acid (about 3000 g) in approximately 750 gincrements, keeping temperature below about 30° C. Sodium carbonate(about 660 g) is added and the pH is adjusted to about 10. The resin isreacted with the solution at ambient temperature overnight. The resin iscollected by filtration, washed six times with ddH₂ O (about 10 L each),six times with 10% acetic acid, and ten times with ddH₂ O. Washing iscontinued with ddH₂ O until the pH reached about 6.0 by litmus paper.The CM-Asp chelating resin was suction dried in preparation for metalloading.

EXAMPLE 2

Metal Loading of CM-Asp Chelating Resin

The CM-Asp chelating resin of Example 1 (about 1 L of suction dried bedvolume) is treated with a transition metal ion solution, e.g. 2 L ofeither 200 mM of cobalt chloride hexahydrate, nickel sulfatehexahydrate, copper sulfate pentahydrate, or zinc chloride, according tothe metal ion deserved. The resin is reacted with the 200 mM metalsolution at room temperature for approximately 72 hours and thencollected by filtration. The metal loaded CM-Asp chelating resin iswashed five times with ddH₂ O (about 1 L each), two times with 100 mMNaCl (about 1 L each), six times with ddH₂ O (about 1 L each), and oncewith 20% aq. ethanol (about 1 L). The resin can be stored in 20% aq.ethanol.

EXAMPLE 3

Comparison of Co²⁺ CM-Asp Resin With Ni²⁺ -NTA on Recombinant 6XHisPrepro-α-Factor Under Denaturing Conditions Using pH Gradient

For a qualitative comparison of the purification of Co²⁺ CM-Aspchelating resin and Ni-NTA under denaturing conditions, the C-terminal,6XHis-tagged prepro-α-factor of S. cerevisiae was expressed in E. coli.One gram bacterial cell pellet was lysed in 6M guanidinium-HCl (Gu-HCl)and 0.1M NaH₂ PO₄, pH 8.0. Three milliliters of clarified lysate wasloaded onto a Co²⁺ CM-Asp chelating resin gravity flow column. Theresin-proteins mixture was washed with 8M urea, 0.1M NaH₂ PO₄, pH 8.0,and eluted with 8M urea, 0.1M NaH₂ PO₄ at three different pHs, 6.3, 5.9,and 4.5. Finally, all bound proteins were eluted with 6M Gu-HCl, 0.1MNaH₂ PO₄, 0.2M acetic acid. Samples from each step were loaded on a 12%polyacrylamide/SDS gel, electrophoresed, and the gel was stained withCoomassie blue. The 6XHis-tagged prepro-a-factor was eluted at pH 6.3 asa single prominent band on the gel.

In the same manner, 3 ml of clarified lysate was loaded onto a Ni-NTAgravity flow column. The resin-proteins mixture was washed and elutedthe same as above. Samples from each step were loaded on a 12%polyacrylamide/SDS gel, electrophoresed, and the gel was stained withCoomassie blue. There were more than 10 protein bands in elution at pH6.3. The 6XHis-tagged prepro-α-factor was a minor band among them. Themajority of the protein was eluted at pH 4.5 without any othercontaminant proteins. This demonstrated that the highly purified6XHis-tagged prepro-α-factor was eluted from Co²⁺ CM-Asp chelating resinat the conditions (pH 6.3) under which Ni-NTA was still releasingcontaminants. The affinity of Co²⁺ CM-Asp chelating resin to6XHis-tagged prepro-α-factor was more selective than Ni-NTA to theprotein.

Results show that highly purified 6XHis-tagged protein elutes from Co²⁺CM-Asp chelating resin while Ni-NTA is still releasing contaminants. SeeFIGS. 2A-2B: FIG. 2A: Results after using 1 ml of Co²⁺ CM-Asp chelatingresin. FIG. 2B: Results after using 1 ml of nickel-NTA.

EXAMPLE 4

Recombinant 6XHis DHFR Purification with CM-Asp Resin Under NativeConditions

N-terminal, 6XHis-tagged mouse dihydrofolate reductase (DHFR, MW 20.3kDa) was expressed in E. coli cells. Cells were then pretreated with0.75 mg/ml lysozyme and disrupted in lysis buffer (100 mM NaH₂ PO₄, 10mM Tris-HCl, pH 8.0) by mechanical shearing, 800 μl of the clarifiedlysate was applied to 100 μl of Co²⁺ CM-Asp chelating resin,pre-equilibrated with lysis buffer, and washed with one ml of lysisbuffer three times. All bound protein was eluted by 300 μl of 100 mMEDTA, pH 8.0. Twenty microliters of lysate and 40 μl of each subsequentfraction from elution were run on a 12% polyacrylamide/SDS gel. The gelwas stained with Coomassie blue. One single protein band was shown at aposition of MW 20.3 kDa. Results showed the selective binding affinityof Co²⁺ CM-Asp chelating resin to 6XHis-tagged DHFR under nativepurification conditions. No discernable binding of host proteinsoccurred.

Results show that Co²⁺ CM-Asp chelating resin has selective bindingaffinity for 6X Histidines. No discernable binding of host proteinsoccurred. See FIG. 3.

EXAMPLE 5

Recombinant 6XHis DHFR Purification with CM-Asp Resin Under DenaturingConditions

N-terminal 6XHis-tagged mouse DHFR was expressed in a 25-ml culture ofE. coli. Cells were pelleted, resuspended in lysis buffer (100 mM NaH₂PO₄, 10 mM Tris-HCl, 8M urea, pH 8.0), and disrupted by vortexing. Sixhundred microliters of clarified lysate were applied to a Co²⁺ CM-Aspchelating resin spin column containing 0.5 ml of Co²⁺ CM-Asp chelatingresin-NX metal affinity resin and centrifuged for 2 minutes at 2,000×g.The column was washed twice with 1 ml of wash buffer (100 mM NaH₂ PO₄,10 mM PIPES, pH 7.0), and bound proteins were eluted with 600 μl ofelution buffer (20 mM PIPES, 100 mM NaCl, 8M urea, pH 6.0). Twentymicroliters of lysate and 40 μl of each subsequent fraction from theelution were loaded onto a 12% polyacrylamide/SDS gel andelectrophoresed. The gel was stained with Coomassie blue. One singleprotein band was shown at the position of 20.3 kDa. Results showed theselective binding affinity of Co²⁺ CM-Asp chelating resin to6XHis-tagged DHFR under denaturing conditions. The binding properties ofCo²⁺ CM-Asp chelating resin to 6X histidines allow proteins eluted undermild pH conditions (pH 6.0) that protect protein integrity.

Results show that bound protein can be eluted at mild pH (pH 6.0). Thisindicates that the binding properties of Co²⁺ CM-Asp chelating resinallow protein elution under mild pH conditions that protect proteinintegrity. See FIG. 4.

EXAMPLE 6

Recombinant 6XHis DHFR Purification with CM-Asp Resin Under NativeConditions with Increasing Concentrations of Beta-Mercaptoethanol

N-terminal, 6XHis-tagged mouse DHFR was expressed in E. coli.Twenty-five milliliters of cell culture were disrupted in 2 ml ofsonication buffer (100 mM NaH₂ PO₄, 10 mM Tris-HCl, and 100 mM NaCl, pH8.0) by freezing and thawing. Then, 2.66 ml of clarified lysate wereapplied to a 200-μl Co²⁺ CM-Asp chelating resin gravity flow column,pre-equilibrated with the sonication buffer. The proteins/resin mixtureswere washed three times with sonication buffer, pH 8.0. All boundproteins were eluted with 600 μl of 100 mM EDTA, pH 8.0. To test theeffect of β-mercaptoethanol on the Co²⁺ CM-Asp chelating resinpurification under native conditions, all buffers used here (containedeither 0, 10, 20, or 30 mM β-mercaptoethanol. Samples from each elutionwere electrophoresed on a 12% polyacrylamide/SDS gel, and the gel wasstained with Coomassie blue. One single protein band at the position ofMW 20.3 kDa was shown from all elutions. The presence ofβ-mercaptoethanol did not obsolete the purity of 6XHis-tagged DHFRpurified by Co²⁺ CM-Asp chelating resin. With up to 30 mMβ-mercaptoethanol in all purification buffers, there was no predominantband at 20.3 kDa in flowthroughs, indicating that no loss of metaloccurred during protein purification using Co²⁺ CM-Asp chelating resinin the presence of β-mercaptoethanol.

Results show that with up to 30 mM β-mercaptoethanol in the purificationbuffer, there is no predominant band at 20.3 kDa in the flowthrough,indicating that no loss of metal occurred during protein purificationusing Co²⁺ CM-Asp chelating resin in the presence of β-mercaptoethanol.See FIG. 5.

EXAMPLE 7

Yields of 6XHis DHFR From Cell Lysates Purified by CM-Asp Versus Ni-NTAin the Presence of Beta-Mercaptoethanol

N-terminal, 6XHis-tagged DHFR was expressed and purified by Co²⁺ CM-Aspchelating resin under native conditions as described in Example 6.Protein concentrations were determined by Bradford assay. Yields wereexpressed as a percentage of total protein in the cell lysate. Theyields of purified 6XHis-tagged DHFR were 14%, 28%, 34%, and 35%respectively, with β-mercaptoethanol present in purification buffers atthe concentrations of 0, 10, 20, and 30 mM. The protein was purified byNi-NTA under the same native conditions; the yields of purified6XHis-tagged DHFR were 4%, 8.8%, 3.4%, and 4% respectively withβ-mercaptoethanol present in purification buffers at the concentrationsof 0, 10, 20, and 30 mM. The yields of purified 6XHis-tagged DHFR weresignificantly higher when using Co²⁺ CM-Asp chelating resin compared toNi-NTA under native purification conditions with β-mercaptoethanol. Thisindicates that the metal ion on Co²⁺ CM-Asp chelating resin is stronglyanchored to SEPHAROSE beads by a CM-ASP metal chelator that is ideal forbinding octahedral metals.

Results show that the yields of purified 6XHis DHFR are significantlyhigher at 10, 20, and 30 mM β-mercaptoethanol using Co²⁺ CM-Aspchelating resin compared to using Ni-NTA. This indicates that the metalion on Co²⁺ CM-Asp chelating resin is strongly anchored to sepharosebeads by CM-Asp metal chelator that is advantageous for bindingoctahedral metals. See FIG. 6.

EXAMPLE 8

Purification of 6XHis GFP by Co²⁺ -CM-Asp Under Native Conditions

N-terminal, 6XHis-tagged green fluorescent protein (GFP) was expressedin E. coli cells. Cells were pelleted, resuspended in sonication buffer(100 mM NaH₂ PO₄, 10 mM Tris-HCl, and 100 mM NaCl, pH 8.0), anddisrupted by freezing and thawing three times. Two milliliters ofclarified lysate were applied to 400 μl of Co²⁺ CM-Asp chelating resin,pre-equilibrated with sonication buffer, and washed three times with 2ml of sonication buffer, pH 8.0. The 6XHis-tagged GFP was eluted with400 μl of 75 mM imidazole buffer containing 20 mM Tris-HCl and 100 mMNaCl, pH 8.0. Samples from each purification step were loaded onto a 12%polyacrylamide/SDS gel, electrophoresed, and the gel was stained withCoomassie blue. One single band was shown at the position of MW 27.8 kDain the elution with 75 mM imidazole. This demonstrated that 6XHis-taggedGFP selectively bound on Co²⁺ CM-Asp chelating resin and can be elutedwith low concentration of imidazole under native purificationconditions.

Samples from each purification step were also loaded on a 12%polyacrylamide/SDS gel, electrophoresed, and transblotted to a PVDFmembrane. The proteins on the blot were probed with anti-GFP monoclonalantibody. One single GFP band was clearly shown in the samples of celllysate and elution. There was no GFP band shown in flowthrough, whichindicated that all expressed GFP in cell lysate was bound to Co²⁺ CM-Aspchelating resin.

Results show that (FIG. 7A) Coomassie blue stained gel shows one singleband in 75 mM imidazole elution. This; indicates that 6X Histidinesselectively bound on Co²⁺ CM-Asp chelating resin. Western analysis datashows no GFP in flowthrough which indicates the high affinity betweenCo²⁺ CM-Asp chelating resin and 6X histidines (FIG. 7B).

EXAMPLE 9

Biological Activity of 6XHis GFP Purified by Co²⁺ CM-Asp

N-terminal, 6XHis-tagged GFP was expressed in E. coli. Cell lysate wasprepared as described in Example 6. The cell lysate was loaded onto a2-ml Co²⁺ CM-Asp chelating resin Disposable Gravity Column, and purifiedusing the Batch/Gravity Flow column purification method as described inExample 8. The column was washed with sonication buffer three times andeluted with 100 mM EDTA, pH 8.0. Samples were collected in microfugetubes from each purification step. The fluorescence of all collectedsamples was detected using an UltraLum Electronic U.V. Transilluminator.Samples of cell lysate and elution showed strong fluorescence. Thisexperiment demonstrated that 6XHis-tagged GFP can be purified tohomogeneity by Co²⁺ CM-Asp chelating resin under native conditions andmaintains biological activity.

The photo of samples from each purification step shows that GFP can bepurified to homogeneity by Co²⁺ CM-Asp chelating resin under nativeconditions, and the fluorescence indicates that GFP purified by Co²⁺CM-Asp chelating resin still maintains its biological activity. See FIG.8.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

We claim:
 1. An immobilized metal ion affinity chromatographypurification method for purification of a recombinant protein, saidmethod comprising:(a) providing carboxymethylated aspartate ligandcomplexed with a transition metal ion in a 2⁺ oxidation state, having acoordination number of 6; (b) loading a mixture of cell lysatecomprising a recombinant protein having a polyhistidine tail to bindwith said ligand: and (c) eluting said recombinant protein with asuitable elutant to obtain a purified recombinant protein, wherein saidtransition metal-complexed carboxymethylated aspartate ligand forms acarboxymethylated aspartate chelating matrix which comprises saidtransition metal and a polymer matrix, wherein said carboxymethylatedaspartate chelating matrix has the structure ##STR2## wherein:M=transition metal ion in a 2⁺ oxidation state with a coordinationnumber of 6; R₁ =a linking arm connecting the nitrogen atom of CM-Aspwith R₂ ; R₂ =a functional linking group through which CM-Asp linkingarm R₁ is connected to R₃ ; and R₃ =a polymer matrix.
 2. An immobilizedmetal ion affinity chromatography complex, wherein said complex has thestructure: ##STR3## wherein: M=transition metal ion in a 2⁺ oxidationstate with a coordination number of 6;R₁ =a linking arm connecting thenitrogen atom of CM-Asp with R₂ ; R₂ =a functional linking group throughwhich CM-Asp linking arm R₁ is connected to R₃ ; and R₃ =a polymermatrix.
 3. The complex, according to claim 2, whereinM=Fe²⁺, Co²⁺, Ni²⁺,Cu²⁺, or Zn²⁺ ; R₁ =--CH₂ CH(OH)CH₂ -- or --CH₂ (OH)CH₂ --O--CH₂CH(OH)CH₂ --; R₂ =O, S, or NH; and R₃ =agarose.
 4. The complex,according to claim 3, whereinM=Co²⁺ ; R₁ CH₂ CH(OH)CH₂ ; R₂ =O; and R₃=agarose, cross-linked.
 5. The method, according to claim 1,whereinM=Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺ ; R₁ =--CH₂ CH(OH)CH₂ -- or--CH₂ (OH)CH₂ --O--CH₂ CH(OH)CH₂ --; R₂ =O, S, or NH; and R₃ =agarose.6. The method, according to claim 5, whereinM=Co²⁺ ; R₁ =CH₂ CH(OH)CH₂ ;R₂ =O; and R₃ =agarose, cross-linked.